The present invention relates to a method of recovering enzymatically active urokinase and prourokinase from a solution, such as a culture medium or cell extract, using a chromatography resin. The invention is based on the discovery that a type of resin developed originally to selectively bind immunoglobulins binds prourokinase and urokinase and can be selectively eluted, and therefore is useful for the commercial, large scale purification of prourokinase and urokinase from solutions containing these proteins.
Urokinase is a useful fibrinolytic agent for initiating the cascade mechanism which disrupts potentially fatal thrombosis. Urokinase is a two-chain glycoprotein having a molecular weight of approximately 32,000-34,000 daltons and a specific activity of about 250,000 CTA units per mg protein. It is an enzymatically active cleavage product of enzymatically inactive prourokinase. Prourokinase is a single chain glycoprotein having a molecular weight of approximately 45,000 55,000 daltons. Unlike urokinase, prourokinase is not active, but is specific for the site of the thrombus. Plasmin cleaves prourokinase to enzymatically active urokinase at that site.
Both forms have been isolated from the urine in a number of ways. One method includes treating the urine with a reagent (e.g., bentonite or other aluminum silicates) which cause the formation of urokinase-containing precipitates, and then eluting the urokinase from the precipitates. Other procedures include bringing the urine in contact with an absorbent reagent and subsequently eluting the absorbent. Known absorbent materials used for this purpose have included, for example, calcium carbonate, barium sulfate, aluminum oxide, calcium phosphate, zinc hydroxide, activated carbon, and hydrated aluminum silicates. Various cation exchange resins have also been useful for this purpose (e.g., U.S. Pat. Nos. 2,983,647; 2,989,440).
Exclusion chromatographic procedures have been practiced which have included the use of, for example, a DEAE cellulose resin or cross-linked dextran gels of the methacrylic-carboxylic acid type (e.g., U.S. Pat. No. 3,256,158 In such a procedure, the resin binds impure proteins and pyrogens, leaving the urokinase in solution.
More recently, affinity chromatographic methods have been practiced using various reagents having an affinity for urokinase bound or adhered to a water-insoluble solid support such as diatomaceous earth, agarose, cellulose, collagen, and other adsorbents. The affinity reagents include basic amino acids, agmatine, aprotinin, derivatives of guanidine and adenine, fibrin, and antibodies which recognize urokinase among many others.
Purification has also been attempted by various electrophoretic and HPLC means.
In an attempt to obtain clinically valuable amounts of urokinase compounds, cells which are known to produce these proteins (e.g., kidney and lung) have been cultured in vitro. Such cells include human and green or Rhesus monkey embryonic and adult kidney, adult and embryonic lung, adult and embryonic heart, placenta, adult thyroid, spleen, and ureter. However, the yield obtained from the culture media of these cells has still not been great, despite attempts to increase output by enriching the culture media with inducers (e.g., various amino acids, saccharides, hormones, fumaric acid, malic acid succinic acid, and/or glycolic acid), or to decrease proteolysis and denaturation (with, e.g., metal chelators, pronase, and trypsin inhibitors). Further attempts to increase the urokinase production of cultured cells were made by culturing urokinase-producing lymphoid hybridoma cells previously transplanted to and propagated in a non-human, warm-blooded animal in the presence of a urokinase inducer (U.S. Pat. No. 4,537,852). However, this process is expensive and time-consuming to practice.
With the advent of recombinant DNA technology, cell lines which have been genetically engineered to produce large amounts of prourokinase have been developed and cultured. These cell lines include microorganisms such as E. coli, yeast, Bacillus, and Neurospora, and mammalian cell lines including monkey and human kidney cells and fibroblasts. The expressed prourokinase must then be recovered in active form from the cell extract or liquid growth media into which it has been secreted. Recovery has been accomplished using essentially the same methodologies as described above for purification from urine.
Recovery from culture media is a significant undertaking in that such media typically contain many other unrelated proteins, some of which have proteolytic activity. For example, serum-supplemented media are known to contain plasmin and other serum proteases which readily degrade prourokinase. Most known purification procedures do not adequately protect urokinase compounds from proteolytic degradation, despite attempts to do so by the addition of metal chelators and various protease inhibitors. As described above, these precautions offer at best only partial protection. Accordingly, known recovery methods are most effective when used to isolate urokinase compounds from serum-free solutions, despite the fact that they are inefficient, and may introduce potentially toxic elements. In addition, purification procedures employing immunoaffinity chromatography may be quite costly when scaled up to meet commercial needs.
Therefore, for commercial quantities of urokinase compounds to be produced in an enzymatically active form, large scale purification procedures are required which effectively, efficiently, and rapidly recover it from the media before much of it is degraded.
Accordingly, it is an object of the invention to provide a rapid, simple, and commercially viable method of isolating a urokinase compound from a urokinase compound-containing solution. Another object is to provide a method of purifying urokinase compounds from culture media. Yet another object is to provide a method of recovering undegraded urokinase compounds substantially free of other non-related proteins from a urokinase compound-containing solution. It is also an object of the invention to provide a method of separating urokinase compounds from other proteins present in serum-supplemented media. It is a further object to provide a method of recovering undegraded urokinase compounds in amounts useful for the large scale commercial production of pharmaceutical formulations containing the same.